In this application the interferon nomenclature announced in Nature, 286, p. 110 (July 10, 1980) is used. E.g., leukocyte interferon is designated IFN-.alpha..
Two classes of interferons ("IF") are known to exist. Interferons of Class I are small, acid stable (glyco)-proteins that render cells resistant to viral infection (A. Isaacs and J. Lindenmann, "Virus Interference I. The Interferon", Proc. Royal Soc. Ser. B., 147, pp. 258-67 (1957) and W. E. Stewart, II, The Interferon System, Springer-Verlag (1979) (hereinafter "The Interferon System")). Class II IFs are acid labile. At present, they are poorly characterized. Although to some extent cell specific (The Interferon System, pp. 135-45), IFs are not virus specific. Instead, IFs protect cells against a wide spectrum of viruses.
Two antigenically distinct species of Class I human interferon ("HIF") are known to exhibit IF activity. One IF species (F) is produced in diploid fibroblast cells. Another IF species (Le) is produced together with minor amounts of F IF in human leukocyte and lymphoblastoid cells. Both are heterogeneous in regard to size, presumably because of the carbohydrate moiety. F IF has been extensively purified and characterized (E. Knight, Jr., "Interferon: Purification And Initial Characterization From Human Diploid Cells", Proc. Natl. Acad. Sci. USA, 73, pp. 520-23 (1976)). It is a glyco-protein of 20,000-26,500 molecular weight (J. Wassenbach et al., "Identification Of The Translation Products Of Human Fibroblast Interferon mRNA In Reticulocyte Lysates", Eur. J. Biochem. 98, pp. 1-8 (1979)). Elucidation of its amino acid sequence is in progress. Two distinct genes, one located on chromosome 2, the other on chromosome 5, code for F IF (D. L. Slate and F. H. Ruddle, "Fibroblast Interferon In Man Is Coded By Two Loci On Separate Chromosomes", Cell, 16, pp. 171-80 (1979)). Le IF has likewise been purified and characterized. Two components have been described, one of 21000 to 22000 and the other of 15000 to 18000 molecular weight. The component of lower molecular weight appears to represent the non-glycosylated form (W. E. Stewart, II et al., "Effect of Glycosylation Inhibitors On The Production And Properties Of Human Leukocyte Interferon", Virology, 97, pp. 473-76 (1979); M. Rubinstein et al., "Human Leukocyte Interferon: Production, Purification To Homogeneity And Initial Characterization", Proc. Natl. Acad. Sci. USA, 76, pp. 640-44 (1979); M. Rubenstein et al., "Human Leukocyte Interferon Purified to Homogeneity", Science, 202, pp. 1289-90 (1978); P. J. Bridgen et al., "Human Lymphoblastoid Interferon", J. Biol. CHem., 252, pp. 6585-87 (1977); K. C. Zoon et al., "Purification And Partial Characterization Of Human Lymphoblastoid Interferon", Proc. Natl. Acad. Sci. USA, 76, pp. 5601-05 (1979); and The Interferon System, p. 173 and references cited therein). A portion of the amino acid sequence of Le IF has been determined, i.e., 20 amino acids from the amino terminus of the polypeptide.
The two species of HIF have a number of different properties. For example, anti-human Le IF antibodies are less efficient against F IF and anti-sera to human F IF have no activity against human Le IF (The Interferon System, p. 151) and Le IF displays a high degree of activity in cell cultures of bovine, feline or porcine origin whereas F IF is hardly active in those cells. In addition, the two IFs result from different mRNA species (and therefore presumable different structural genes) that code for polypeptides of different primary sequence (R. L. Cavalieri et al., "Synthesis Of Human Interferon By Xenopus laevis Oocytes: Two Structural Genes For Interferon In Human Cells", Proc. Natl. Acad. Sci. USA, 74, pp. 3287-91 (1977)).
Although both Le and F IFs occur in a glycosylated form, removal of the carbohydrate moiety (P. J. Bridgen et al., supra) or synthesis of IF in the presence of inhibitors which preclude glycosylation (W. E. Stewart, II et al., Virology, supra; J. Fujisawa et al., "Nonglycosylated Mouse L Cell Interferon Produced By The Action Of Tunicamycin", J. Biol. Chem., 253, pp. 8677-79 (1978)) yields a smaller form of IF which still retains most or all of its IF activity.
Both F IF and Le IF may, like many human proteins, be polymorphic. Therefore, cells of particular individuals may produce IF species within each of the more general F IF and Le IF classes which are physiologically similar but structurally slightly different than the class of which it is a part. Therefore, while the protein structure of the F IF or Le IF may be generally well-defined, particular individuals may produce IFs that are slight variations thereof.
IF is usually not detectable in normal or healthy cells (The Interferon System, pp. 55-57). Instead, the protein is produced as a result of the cell's exposure to an IF inducer. IF inducers are usually viruses but may also be non-viral in character, such as natural or synthetic double-stranded RNA, intracellular microbes, microbial products and various chemical agents. Numerous attempts have been made to take advantage of these non-viral inducers to render human cells resistant to viral infection (S. Baron and F. Dianzani (eds.), Texas Reports On Biology And Medicine, 35 ("Texas Reports"), pp. 528-40 (1977)). These attempts have not been very successful. Instead, use of exogenous IF itself is now preferred.
As an antiviral agent, HIF has been used to treat the following: respiratory infections, (Texas Reports, pp. 486-96); herpes simplex keratitis (Texas Reports, pp. 497-500); acute hemorrhagic conjunctivitis, (Texas Reports, pp. 501-10); varicella zoster, (Texas Reports, pp. 511-15); cytomegalovirus infection (Texas Reports, pp. 523-27); and hepatitis B, (Texas Reports, pp. 516-22). See also The Interferon System, pp. 307-19. However, large scale use of IF as an antiviral agent requires larger amounts of HIF than heretofore have been available.
IF has other effects in addition to its antiviral action. For example, it antagonizes the effect of colony stimulating factor, inhibits the growth of hemopoietic colony-forming cells and interferes with the normal differentiation of granulocyte and macrophage precursors (Texas Reports, pp. 343-49). It also inhibits erythroid differentiation in DMSO-treated Friend leukemia cells (Texas Reports, pp. 420-28). IF may also play a role in regulation of the immune response. Depending upon the dose and time of application in relation to antigen, Le IF can be both immunopotentiating and immunosuppressive in vivo and in vitro (Texas Reports, pp. 357-69). In addition, specifically sensitized lymphocytes have been observed to produce IF after contact with antigen. Such antigen-induced IF could therefore be a regulator of the immune response, affecting both circulating antigen levels and expression of cellular immunity (Texas Reports, pp. 370-74). IF is also known to enhance the activity of killer lymphocytes and antibody-dependent cell-mediated cytotoxicity (R. R. Herberman et al., "Augmentation By Interferon Of Human Natural And Antibody Dependent Cell-Mediated Cytotoxicity", Nature, 277, pp. 221-23 (1979); P. Beverley and D. Knight, "Killing Comes Naturally", Nature, 278, pp. 119-20 (1979); Texas Reports, pp. 375-80). Both of these species are probably involved in the immunological attack on tumor cells.
Therefore, in addition to its use as a human antiviral agent, HIF has potential application in antitumor and anticancer therapy (The Interferon System, pp. 319-21). It is now known that IFs affect the growth of many classes of tumors in many animals (The Interferon System, pp. 292-304). They, like other antitumor agents, seem most effective when directed against small tumors. The antitumor effects of animal IF are dependent on dosage and time but have been demonstrated at concentrations below toxic levels. Accordingly, numerous investigations and clinical trials have been and continue to be conducted into the antitumor and anticancer properties of HIFs. These include treatment of several malignant diseases such as osteosarcoma, acute myeloid leukemia, multiple myeloma and Hodgkin's disease (Texas Reports, pp. 429-35). Although the results of these clinical tests are encouraging, the antitumor and anticancer applications of HIF have been severely hampered by lack of an adequate supply of purified HIF.
At the biochemical level IFs induce the formation of at least 3 proteins, a protein kinase (B. Lebleu et al., "Interferon, Double-Stranded RNA And Protein Phosphorylation", Proc. Natl. Acad. Sci. USA, 73, pp. 3107-11 (1976), A. G. Hovanessian and I. M. Kerr, "The (2'-5') Oligoadenylate (ppp A2'-5'A2'-5'A) Synthetase And Protein Kinase(s) From Interferon-Treated Cells", Eur. J. Biochem., 93, pp. 515-26 (1979)), a (2'-5')oligo(A) polymerase (A. G. Hovanessian et al., "Synthesis Of Low-Molecular Weight Inhibitor Of Protein Synthesis With Enzyme From Interferon-Treated Cells", Nature, 268, pp. 537-39 (1977), A. G. Hovanessian and I. M. Kerr, Eur. J. Biochem., supra) and a phosphodiesterase (A. Schmidt et al., "An Interferon-Induced Phosphodiesterase Degrading (2'-5')oligoisoadenylate And The C-C-A Terminus of tRNA", Proc. Natl. Acad. Sci. USA, 76, pp. 4788-92 (1979)). The appearance of these enzymes in cells treated with IF should allow a further characterization of proteins with IF-like activity.
Today, human leukocyte IF is produced either through human cells grown in tissue culture or through human leukocytes collected from blood donors. 2.6.times.10.sup.9 IU of crude IF have been reported from 800 l of cultured Namalva cells (P. J. Bridgen et al., supra). At very large blood centers, e.g., the Finnish Red Cross Center in Helsinki, Finland, the production capacity is about 10.sup.11 IU annually. Since dosage is typically 3.times.10.sup.6 IU per patient per day, neither of these sources are adequate to provide the needed commercial quantities of HIF. Therefore, production of IF by other procedures is attractive.
The crude IF produced as above may be purified to higher specific activity. For example, in yields of about 25%, Le IF isolated from blood cells has been purified to about 10.sup.9 IU/mg (L. S. Lin et al., "Purification Of Human Leukocyte Interferon To Apparent Homogeneity: Criteria For Purity", Abs. Ann. Meeting Amer. Soc. Microbiol. (1978) and The Interferon System, pp. 156-71). Because the specific activity of IF is so high, in the order of 4.0.times.10.sup.8 -10.sup.9 IU/mg, the amount of IF protein required for commercial applications is low. For example, 100 grams of pure IF would provide between 3 and 30 million doses.
Recent advances in molecular biology have made it possible to introduce the DNA coding for specific nonbacterial eukaryotic proteins into bacterial cells. In general, with DNA other than that prepared via chemical synthesis, the construction of such recombinant DNA molecules comprises the steps of producing a single-stranded DNA copy (cDNA) of a purified messenger RNA (mRNA) template for the desired protein; converting the cDNA to double-stranded DNA; linking the DNA to an appropriate site in an appropriate cloning vehicle to form a recombinant DNA molecule and transforming an appropriate host with that recombinant DNA molecule. Such transformation may permit the host to produce the desired protein.
Several non-bacterial proteins and genes have been obtained in E. coli using recombinant DNA technology. These include a protein displaying rat proinsulin antigenic determinants (L. Villa-Komaroff et. al., "A Bacterial Clone Synthesizing Proinsulin", Proc. Natl. Acad. Sci. USA, 75, pp. 3727-31 (1978)), rat growth hormone (P. H. Seeburg et al., "Synthesis Of Growth Hormone By Bacteria", Nature, 276, pp. 795-98 (1978)), mouse dihydrofolate reductase (A. C. Y. Chang et al., "Phenotypic Expression in E. coli Of A DNA Sequence Coding For Mouse Dihydrofolate Reductase", Nature, 275, pp. 617-24 (1978)), human somatostatin (K. Itakura et al., "Expression in Escherichia coli Of A Chemically Synthesized Gene For The Hormone Somatostatin", Science, 198, pp. 1056-63 (1977)); European patent applications 0,001,929, 0,001,930, and 0,001,931 and cognate applications in other countries), the A and B polypeptide chains of human insulin (D. V. Goeddel et al., "Expression in Escherichia coli Of Chemically Synthesized Genes For Human Insulin", Proc. Natl. Acad. Sci. USA, 76, pp. 106-10 (1979) and the European and related patent specifications, supra), antigens of human hepatitis B virus (C. J. Burrell et al., "Expression in Escherichia coli: Of Hepatitis B Virus DNA Sequences Cloned In Plasmid pBR322", Nature, 279, pp. 43-7 (1979) and M. Pasek et al., "Hepatitis B Virus Genes And Their Expression In E. coli", Nature, 282, pp. 575-79 (1979)), human growth hormone (D. V. Goeddel et al., "Direct Expression In Escherichia coli Of A DNA Sequence Coding For Human Growth Hormone", Nature, 281, pp. 544-51 (1979)), and SV40 t antigen (T. M. Roberts et al., "Synthesis Of Simian Virus 40 t Antigen In Escherichia coli", Proc. Natl. Acad. Sci. USA, 76, pp. 5596-600 (1979)).
In addition, at least in the case of ovalbumin DNA, it is known that appropriate fusion of the particular DNA to a strong bacterial promoter or expression control sequence produces larger amounts of the desired ovalbumin-like protein, i.e., about 0.5 to 1% of the total protein mass of an E. coli cell (O. Mercereau-Puijalon et al., "Synthesis Of An Ovalbumin-Like Protein By Escherichia coli K12 Harboring A Recombinant Plasmid", Nature, 275, pp. 505-10 (1978); T. H. Fraser and B. J. Bruce, "Chicken Ovalbumin Is Synthesized And Secreted By Escherichia coli", Proc. Natl. Acad. Sci. USA, 75, pp. 5936-40 (1978)).
None of the foregoing, however is directed, as is this invention, toward the synthesis of HIF with use of recombinant DNA technology. Moreover, the execution of each of the foregoing examples is enhanced by the availability of the sequence information required to prepare a synthetic gene (Itakura et al., supra) or of a cell type or virus rich in a particular DNA sequence (C. J. Burrell et al., supra) or mRNA species (Villa-Komaroff et al., supra) which readily allows preparation and identification of bacterial clones containing the desired hybrid DNA, or of a system allowing the selection of E. coli expressing the desired protein (A.C.Y. Chang et al., supra). No such facilitating circumstances exist in the case of the IF system.